Polarized expression of different potassium channel subtypes on opposite membrane domains of renal collecting duct cells insures an efficient and precisely controlled potassium secretion process, critical for potassium homeostasis. The overarching goal of the present proposal is to develop a mechanistic explanation of the poorly understood trafficking processes that drive polarized localization and physiologically regulate the cell surface density of two closely related channels in the collecting duct (the apical secretory channel, Kir1.1 (ROMK), and a basolateral membrane channel, Kir2.3). The program logically builds on our recent discoveries, defining the trafficking signals in these channels and the elucidating the intracellular sorting and retention machinery that interacts with them. Specifically, we will address the following critical and timely questions: 1. How are the polarized trafficking signals in basolateral membrane Kir channels interpreted? Here, we test a novel mechanism whereby independent signals in Kir2.3 sequentially interact with intracellular sorting machinery and a PDZ scaffold complex, Lin-7/CASK, to drive basolateral-directed traffic in the biosynthetic and endocytotic pathways. 2. Does Lin-7C interaction regulate basolateral Kir channel in the renal cortical collecting duct during potassium adaptation? Collecting duct specific Lin-7C knockout mice will be examined to test the hypothesis that the increase in basolateral membrane conductance in potassium adaptation is influenced by interaction with a specific Lin-7 isoform, Lin-7C. 3. What is the molecular mechanism by which Kir1.1 (ROMK) channel density is controlled by endocytosis. Here we test the hypothesis that a "NPXY"-type signal in Kir1.1 serves as recognition site for binding to a member of a new class of clathrin-adaptor proteins, ARH, and this interaction marks Kir1.1 channels for rapid endocytosis. Moreover, ARH knockout mice will be used to test the hypothesis that physiological attenuation of Kir1.1 channels at the apical membrane activity is influenced by interaction with ARH. In doing so, the program of investigation will provide new insights into the fundamental trafficking mechanisms that underpin potassium secretion in health and to understand what happens when trafficking signals and trafficking machinery goes wrong in disease. Project Narrative: Potassium channels that underpin potassium balance must be precisely organized at two polarized membrane domains in the Kidney for efficient renal potassium secretion. Disruption of ion channel trafficking and surface expression can, in fact, have devastating consequences on salt and mineral balance. Despite its importance, a long-standing and fundamental question in cell biology and physiology has been how the number and location of these membrane proteins are precisely controlled. In the present proposal, we elucidate the molecular mechanisms driving membrane trafficking of these channels in health and study what may happen when these processes go awry in disease. Thus, the studies should provide novel insights into the molecular basis of renal K handling and K homeostasis in health and disease while illuminating fundamental mechanisms of membrane protein targeting in the kidney.